1. Volume 9, Issue 3, Pages 305-313 (September 1998)
Differential TCR Signaling Regulates Apoptosis and Immunopathology during Antigen Responses In Vivo 
Behazine Combadière, Caetano Reis e Sousa, Carol Trageser, Li-Xin Zheng, Charles R Kim, Michael J Lenardo 
Immunity 
Volume 9, Issue 3, Pages (September 1998)
DOI: /S (00)

2. Figure 1 Antigen-Induced Death Is Clonotype Specific
(Aa) Cell death assay using three types of cells: cycling A.E7, CMFDA-labeled P13.9 APC, and d-He-labeled cycling CAB from B10.A mice (Boehme et al. 1995). Cells were incubated for 24 hr in medium alone or in the presence of PCC 88–104 (wt) peptide or immobilized anti-CD3ε MAb (2C11).
(Ab) Cell death assay using two types of cells: cycling CMFDA-loaded A.E7 and Vβ8+ T cell blasts. Cells were incubated with either immobilized anti-CD3ε MAb (2C11) or biotinylated anti-Vβ8 and 1 μg/ml of avidin. Cell loss for each cell type was measured by flow cytometric counting of viable cells.
(B) Expression of CD95/Apo-1/Fas receptor on A.E7 cells or CAB by flow cytometry: control hamster Ig (dotted line) and anti-Fas (solid line). One of three representative experiments is shown.
(C) Dose–response analyses of cycling A.E7 cells stimulated with different doses of PCC (88–104) wt peptide or with variant peptides R99 or A99 at 100 μM. T cell apoptosis was measured after stimulation for 24 hr and after addition of 7 amino-actinomycin D (7-AAD). All error bars shown represent one SD from the mean.
(D) R99 or A99 stimulation render A.E7 susceptible to Fas- or TNFR-mediated apoptosis. Cycling A.E7 cells were stimulated with P13.9 APC preincubated with 100 μM of R99 or A99 in the presence of either medium alone or biotinylated anti-mouse Fas (Jo2, 10 μg/ml), avidin (1 μg/ml), and/or TNFα (50 U/ml). After 24 hr incubation at 37°C, A.E7 cells were stained with fluoresceinated anti-CD4 to differentiate them from APC and cell death was assessed after addition of 7-AAD by flow cytometry.
(E) Cycling AND T cells specific for PCC 88–104 were stimulated with CMFDA-loaded P13.9 APC preincubated with 100 μM of wt, G99, S99, or D99 in the presence of either medium alone or biotinylated anti-mouse Fas (Jo2, 10 μg/ml), avidin (1 μg/ml), and/or TNFα (50 U/ml). T cell apoptosis was measured after 24 hr.
Immunity 1998 9, DOI: ( /S (00) )

3. Figure 2 Variant Ligands R99 and A99 Do Not Induce Death Molecules
(A) Cycling A.E7 cells were stimulated with variants or wt peptide (100 μM) for 12 hr in presence of metalloproteinase inhibitor and were stained with PE-conjugated anti-FasL and FITC-conjugated anti-CD4. Dead cells were gated out and viable CD4+ A.E7 cells were analyzed for FasL expression.
(B) TNFα production in the same experiment was measured by ELISA. Results are representative of at least three independent experiments.
(C) Variant peptides must be presented on APCs to promote cell death. Cycling A.E7 cells were stimulated with either CMFDA-loaded DAP.3 (H-2Ek−) (left side) or P13.9 (H-2Ek+) APC (right side) preincubated with either medium alone (no peptide) or 100 μM of R99 in the presence of cross-linked anti-mouse Fas Ab (5 μg/ml) and/or 20 U/ml of TNFα. Cells were incubated for 24 hr at 37°C. Percent cell death was measured by flow cytometry after the addition of PI. All error bars are shown and represent one SD from the mean.
Immunity 1998 9, DOI: ( /S (00) )

4. Figure 3
Cells that Receive a Competence to Die Signal Undergo Apoptosis in the Presence of Third-Party T Cells Expressing FasL or TNF In Vitro
(A) Scheme of the three-cell system. Activated, cycling AND T cells recognize wt PCC 88–104 and the R99 variant as agonists, up-regulate FasL and TNFα, and die by apoptosis. Shown are the hypothetical interactions between the A.E7 and AND T cells tested in this experiment.
(B) Cycling T cells from AND-TCR transgenic mice and CMFDA-loaded cycling A.E7 cells were added to P13.9 APC preincubated with wt or variant peptides at various concentrations. AND T cells were stained with PE-conjugated anti-H-2Kb prior to flow cytometry. Cell death was measured after the addition of 7-AAD (Sigma Chemical Co., St Louis, MO) by flow cytometry.
(C) To block cell death, Fas-Fc (10 μg/ml) and TNFR-Fc (10 μg/ml) or both were included in the assay described above. Error bars represent one SD from the mean.
Immunity 1998 9, DOI: ( /S (00) )

5. Figure 4
Effect of the A99 and R99 Variant Peptides on A.E7 Cells In Vivo during Immune Responses to an Unrelated Antigen
(A) Experimental scheme. Time course of injections of cells and antigens. The asterisk indicates the time at which death-inducing peptide challenges were given, as indicated in (B). See Experimental Procedures for details.
(B) Spleen cell suspensions were analyzed for the presence of TCR+CMFDA+ T cells. Events (1.5 million) were acquired by flow cytometry after staining of the T cells with PE-conjugated anti-TCRβ. Percent TCR+CMFDA+ A.E7 cell loss was calculated compared to control HEL + PBS injected mice in each group. A fraction of spleen cells was further analyzed for the presence of remaining apoptotic cells by using a TUNEL assay as described by the manufacturer (Trevigen, Gaithersburg, MD).
Immunity 1998 9, DOI: ( /S (00) )

6. Figure 5
The Wt Agonist Peptide, but not Partial Signaling Peptides, Induces Liver Damage
Paraffin sections of liver after hematoxylin and eosin stain (American HistoLabs, Gaithersburg, MD) from the experiment described in Figure 4. (A) Liver section (100× original magnification) from the PBS group injected with A.E7 cells plus either PBS (left panel) or wt PCC 88–104 peptide (right panel), together with HEL 46–61. Liver infarction comprising venous thrombosis and hepatocyte necrosis present throughout the whole liver are seen after wt peptide treatment only and are indicated by an asterisk. (Ba), (Bb), and (Bc) correspond to the PBS group, and (Bd), (Be), and (Bf) to the HEL group. Enlargement of liver sections (630× original magnification) from mice injected with HEL 46–61 plus the indicated PCC peptides: no peptide (Ba and Bd), wt (Bb and Be), and A99 (Bc and Bf). No areas of liver damage were observed after the injection of variant peptide A99 in either group; large areas of liver damage after treatment with wt peptide are indicated by an asterisk. (C) Detection of A.E7 cells by flow cytometric analysis of cell suspensions prepared from the liver. Percent CMFDA+ cells (A.E7) are shown for each plot. (A), (B), and (C) are from the same experiment.
Immunity 1998 9, DOI: ( /S (00) )

7. Figure 6
Variant Peptides that Selectively Promote T Cell Apoptosis through FasL and TNF Do Not Induce Liver Damage In Vivo
(A) Cell suspensions of spleen were analyzed for the presence of TCR+CMFDA+ T cells. Events (1.5 million) were acquired by flow cytometry after staining of the T cells with PE-conjugated anti-TCRβ. Percent TCR+CMFDA+ A.E7 cell loss was calculated and compared to control PBS-injected mice in each group. A fraction of spleen cells was further analyzed for the presence of remaining apoptotic cells by using a TUNEL assay as described by the manufacturer (Trevigen, Gaithersburg, MD). Similar results were obtained in the liver (data not shown).
(B) Liver sections (100× original magnification) from the PBS group injected with A.E7 cells plus either PBS, wt PCC 88–104, Y99, or C99 peptides. Liver infarction comprising venous thrombosis and hepatocyte necrosis that were seen only with wt peptide treatments are indicated by an asterisk and are present throughout the entire liver.
Immunity 1998 9, DOI: ( /S (00) )